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Aug 4, 2017 - Washington State University Tri-Cities,. United States. Reviewed by: Lisa Y. Stein,. University of Alberta, Canada. Mee-Rye Park,. Columbia ...
METHODS published: 04 August 2017 doi: 10.3389/fmicb.2017.01508

AmoA-Targeted Polymerase Chain Reaction Primers for the Specific Detection and Quantification of Comammox Nitrospira in the Environment Edited by: Martin G. Klotz, Washington State University Tri-Cities, United States Reviewed by: Lisa Y. Stein, University of Alberta, Canada Mee-Rye Park, Columbia University, United States Hongkeun Park, Columbia University, United States *Correspondence: Petra Pjevac [email protected] Sebastian Lücker [email protected] Holger Daims [email protected]

These authors have contributed equally to this work.

Specialty section: This article was submitted to Microbiotechnology, Ecotoxicology and Bioremediation, a section of the journal Frontiers in Microbiology Received: 04 April 2017 Accepted: 27 July 2017 Published: 04 August 2017 Citation: Pjevac P, Schauberger C, Poghosyan L, Herbold CW, van Kessel MAHJ, Daebeler A, Steinberger M, Jetten MSM, Lücker S, Wagner M and Daims H (2017) AmoA-Targeted Polymerase Chain Reaction Primers for the Specific Detection and Quantification of Comammox Nitrospira in the Environment. Front. Microbiol. 8:1508. doi: 10.3389/fmicb.2017.01508

Petra Pjevac 1*, Clemens Schauberger 1 † , Lianna Poghosyan 2 † , Craig W. Herbold 1 , Maartje A. H. J. van Kessel 2 , Anne Daebeler 1 , Michaela Steinberger 1 , Mike S. M. Jetten 2 , Sebastian Lücker 2*, Michael Wagner 1 and Holger Daims 1* 1

Division of Microbial Ecology, Department of Microbiology and Ecosystem Science, Research Network ‘Chemistry meets Microbiology’, University of Vienna, Vienna, Austria, 2 Department of Microbiology, Institute for Water and Wetland Research (IWWR), Radboud University, Nijmegen, Netherlands

Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be catalyzed by the concerted activity of ammonia- and nitrite-oxidizing microorganisms. Only recently, complete ammonia oxidizers (“comammox”), which oxidize ammonia to nitrate on their own, were identified in the bacterial genus Nitrospira, previously assumed to contain only canonical nitrite oxidizers. Nitrospira are widespread in nature, but for assessments of the distribution and functional importance of comammox Nitrospira in ecosystems, cultivation-independent tools to distinguish comammox from strictly nitrite-oxidizing Nitrospira are required. Here we developed new PCR primer sets that specifically target the amoA genes coding for subunit A of the distinct ammonia monooxygenase of comammox Nitrospira. While existing primers capture only a fraction of the known comammox amoA diversity, the new primer sets cover as much as 95% of the comammox amoA clade A and 92% of the clade B sequences in a reference database containing 326 comammox amoA genes with sequence information at the primer binding sites. Application of the primers to 13 samples from engineered systems (a groundwater well, drinking water treatment and wastewater treatment plants) and other habitats (rice paddy and forest soils, rice rhizosphere, brackish lake sediment and freshwater biofilm) detected comammox Nitrospira in all samples and revealed a considerable diversity of comammox in most habitats. Excellent primer specificity for comammox amoA was achieved by avoiding the use of highly degenerate primer preparations and by using equimolar mixtures of oligonucleotides that match existing comammox amoA genes. Quantitative PCR with these equimolar primer mixtures was highly sensitive and specific, and enabled the efficient quantification of clade A and clade B comammox amoA gene copy numbers in environmental samples. The measured relative abundances of comammox Nitrospira, compared to canonical ammonia oxidizers, were highly variable across environments. The new comammox amoA-targeted primers enable more encompassing future studies

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of nitrifying microorganisms in diverse habitats. For example, they may be used to monitor the population dynamics of uncultured comammox organisms under changing environmental conditions and in response to altered treatments in engineered and agricultural ecosystems. Keywords: nitrification, comammox, Nitrospira, amoA, marker gene, PCR

INTRODUCTION

related to the unique comammox amoA revealed the presence of putative comammox organisms in various environments including soils (paddy rice soils, other agricultural soils, forest soils, grassland soils), freshwater habitats (wetlands, rivers, lakes, groundwater basins), groundwater wells (GGWs), full-scale wastewater treatment plants (WWTPs), and drinking water treatment plants (DWTPs) (Daims et al., 2015; van Kessel et al., 2015). While this provides strong indications of a broad habitat range of comammox organisms, current knowledge about the environmental distribution and abundance of comammox Nitrospira is very limited and needs to be explored. The amoA genes of comammox Nitrospira form two monophyletic sister clades (Daims et al., 2015; van Kessel et al., 2015), which are referred to as clade A and clade B. Clade A also contains some genes that were previously assigned to the methanotroph Crenothrix polyspora (Stoecker et al., 2006), but this assignment has recently been corrected (Oswald et al., 2017). Established PCR primer sets specifically targeting the amoA genes of AOB or AOA (Sinigalliano et al., 1995; Rotthauwe et al., 1997; Juretschko et al., 1998; Stephen et al., 1999; Nold et al., 2001; Norton et al., 2002; Webster et al., 2002; Okano et al., 2004; Francis et al., 2005; Treusch et al., 2005; Mincer et al., 2007; Junier et al., 2008; Meinhardt et al., 2015) do not amplify any clade A and clade B comammox amoA. Co-amplification of some comammox amoA genes occurs with a primer set targeting both betaproteobacterial amoA and the A subunit of the particulate methane monooxygenase (pmoA of pMMO; Holmes et al., 1995; Luesken et al., 2011). These primers, however, only target a fraction of the known comammox amoA genes. A recently established two-step PCR protocol (Wang et al., 2016) relies on the forward primer published by Holmes et al. (1995) and a new, highly degenerate reverse primer. It detects the bacterial copper-containing monooxygenase (CuMMO) genes including pmoA, betaproteobacterial amoA, and at least the comammox amoA genes that are amplified by the Holmes forward primer. However, because of its broad coverage of the CuMMOs, this primer pair does not allow a specific detection or quantification by quantitative PCR (qPCR) of comammox amoA genes. The two sets of comammox amoA targeted (q)PCR primers recently published by Bartelme et al. (2017) are, in contrast, highly specific. These primers only detect a small fraction of available comammox amoA gene sequences within clade A, and cannot be used to amplify comammox clade B amoA genes. To enable the direct detection and quantification of comammox amoA genes in environmental samples, we designed in this study two new amoA-targeted primer sets specific for clade A or clade B comammox amoA genes, respectively. Subsequently, we applied these new primers to efficiently screen various habitats for the presence of comammox organisms and

Nitrification is an essential process of the global biogeochemical nitrogen cycle and plays a pivotal role in biological wastewater treatment and in drinking water production. The recent discovery of the first complete ammonia oxidizers (“comammox”) in the bacterial genus Nitrospira (Daims et al., 2015; van Kessel et al., 2015) was highly unexpected since Nitrospira were always regarded as canonical nitrite-oxidizing bacteria (NOB) (Watson et al., 1986; Ehrich et al., 1995; Spieck et al., 2006; Lebedeva et al., 2008, 2011). This discovery has raised a number of important questions, such as how often comammox Nitrospira occur in nitrifying microbial communities, and how relevant they are for nitrification compared to ammonia-oxidizing bacteria (AOB), archaea (AOA), and NOB. Members of the genus Nitrospira have been assigned to at least six sublineages (Daims et al., 2001; Lebedeva et al., 2008, 2011). They are widespread in virtually all oxic natural and engineered ecosystems, and an impressively high diversity of coexisting uncultured Nitrospira strains has been detected in wastewater treatment plants and soils (Pester et al., 2014; GruberDorninger et al., 2015). All known comammox organisms belong to Nitrospira sublineage II (Daims et al., 2015; van Kessel et al., 2015; Palomo et al., 2016; Pinto et al., 2016). This sublineage contains also canonical NOB, which lack the genes for ammonia oxidation (Daims et al., 2001; Koch et al., 2015). Moreover, the known comammox Nitrospira do not form a monophyletic clade within Nitrospira lineage II in phylogenies based on 16S rRNA genes or nxrB, the gene encoding subunit beta of the functional key enzyme nitrite oxidoreductase. Instead, they intersperse with the strict NOB in these phylogenetic trees (Daims et al., 2015; van Kessel et al., 2015; Pinto et al., 2016). Finally, it remains unknown whether other Nitrospira sublineages (Daims et al., 2001; Lebedeva et al., 2008, 2011) also contain comammox members. Consequently, it is impossible to infer from 16S rRNA or nxrB gene phylogenies whether yet uncharacterized Nitrospira bacteria are comammox or strict NOB, although such attempts have been published (Gonzalez-Martinez et al., 2016). Intriguingly, comammox Nitrospira possess novel types of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO), the key enzymes of aerobic ammonia oxidation. The comammox AMO is phylogenetically distinct from the AMO forms of canonical AOB and AOA (Daims et al., 2015; van Kessel et al., 2015). The amoA gene encoding AMO subunit A has become a widely used functional and phylogenetic marker gene for bacterial and archaeal ammonia oxidizers (Rotthauwe et al., 1997; Purkhold et al., 2000; Junier et al., 2008; Pester et al., 2012). Public database mining for sequences

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to rapidly quantify the abundances of comammox in selected samples.

DNA extracted by a phenol-chloroform based method (details below) from a GWW sample (Wolfenbüttel, Germany), which contained both clade A and clade B comammox Nitrospira (Daims et al., 2015). An annealing temperature range from 42 to 52◦ C was chosen for experimental evaluation based on the theoretical melting temperature of 48◦ C of the designed primers (http://biotools.nubic.northwestern.edu/OligoCalc. html). Thermal cycling was carried out with an initial denaturation step at 94◦ C for 5 min, which was followed by 25 cycles of denaturation at 94◦ C for 30 s, primer annealing at 42–52◦ C for 45 s, and elongation at 72◦ C for 1 min. Cycling was completed by a final elongation step at 72◦ C for 10 min. The optimal annealing temperature for both primer pairs was found to be 52◦ C. At lower annealing temperatures, unspecific amplification of DNA fragments shorter than the expected amplicon length (415 bp) was observed. Since the amplification efficiency of correctly sized amplicons was lower at 52◦ C than at 48–50◦ C, temperatures above 52◦ C were not evaluated. The PCR reactions were performed in a DreamTaq Green PCR mix with 1 × Dream Taq Green Buffer containing 2 mM MgCl2, 0.025 U DreamTaq DNA polymerase, 0.2 mM dNTPs, 0.5 µM primers and 0.1 mg/mL bovine serum albumin (Fermentas, Thermo Fischer Scientific, Waltham, MA, USA).

MATERIALS AND METHODS Database Mining and Sequence Collection The amino acid sequences of bacterial AmoA and PmoA were extracted from publicly available metagenomic datasets stored in the Integrated Microbial Genomes databases (IMG-ER and -MER) using the functional profiler tool against a specific bacterial AmoA/PmoA pfam (PF02461). For characterization of comammox AmoA, betaproteobacterial AmoA, and PmoA, sequences collected from the Integrated Microbial Genomes databases were augmented with nearly full length amino acid sequences collected from the Pfam site and from NCBI Genbank as described in Daims et al. (2015). Collected sequences were filtered against a hidden Markov model (hmm) using hmmsearch (http://hmmer.janelia.org/) with the AmoA/PmoA hmm (PF02461) requiring an expect value of